In biotechnology, separation and analysis of biological samples is critically important. Moreover, it is desirable to conduct multiple separations and analyses of the separated components simultaneously to increase the speed and efficiency at which chemical samples are evaluated. For example, separation technologies such as electrophoresis are used in DNA sequencing, protein molecular weight determination, genetic mapping, and other types of processes used to gather large amounts of analytical information about particular chemical samples.
One method used to separate chemical samples into their component parts is electrophoresis. Electrophoresis is the migration of charged colloidal particles or molecules through a solution under the influence of an applied electric field usually provided by immersed electrodes, where the colloidal particles are a suspension of finely divided particles in a continuous media. Historically, a polymer gel containing the finely divided particles is placed between two glass plates and an electric field applied to both ends of the plates. This method, however, offers a low level of automation and long analysis times.
More recently, the capillary electrophoresis method was developed, which has the added advantages of speed, versatility and low running costs. Operation of a capillary electrophoresis system involves application of a high voltage (typically 5–15 kV) across a narrow bore capillary (typically 25–100 μm). The capillary is filled with electrolyte solution that conducts current through the inside of the capillary. The ends of the capillary are placed in reservoirs filled with the electrolyte. Electrodes made of an inert material such as platinum are also inserted into the electrolyte reservoirs to complete the electrical circuit. A small volume of sample is injected into one end of the capillary. Application of a voltage across the capillary causes movement of the sample ions towards a corresponding electrode at the opposite end of the capillary. Depending upon various factors, the different components in the sample will travel through the capillary at different rates. Therefore, the sample will be separated into its various components or at least into those components that travel through the capillary at the same rate. A detector, such as a emission detector, is positioned at the opposite end of the capillary to detect the presence of the various sample components as they travel through the capillary and past the detector. The sample may be labeled with a fluorescent marker so that when the sample components pass through a beam of light at the detector, the fluorescent marker fluoresces, and the fluorescence is detected as an electric signal. The intensity of the electric signal depends on the amount of fluorescent marker present in the detection zone. The plot of detector response with time is then generated which is termed an electropherogram.
In traditional capillary electrophoresis systems, analysis or detection of the separated components is performed while the sample is still located within the capillary and may be accomplished using photometric techniques such as absorbance and fluorescence. Absorbance and fluorescence is where excitation light is directed toward the capillary tube, and light emitted from the sample (e.g., fluorescence) is measured by a detector, thereby providing information about the separated components. Therefore, in these systems, excitation light directed at the sample, as well as light emitted from the sample, must be transmitted through the capillary's walls. A drawback of this approach is that the tubular shape of the fused silica capillaries causes significant scattering of light. The problem associated with light scattering is exacerbated by the fact that silica itself gives off a background level of fluorescence and further by having multiple capillaries disposed side-by-side, as scattered excitation light from one capillary interferes with the detection of samples in neighboring capillaries.
A preferred variation of the capillary electrophoresis system described above replaces the capillary tubes with a number of parallel channels formed in a substrate such as a plate or chip, where the channels are in fluid communication with a pair of electrodes. This type of system is known, for example, as a microfluidic chip or micro-channel array. Such microfluidic chips are advantageous for high-throughput applications where a large number of samples are to be separated or otherwise manipulated at one time. However, similar to the traditional capillary electrophoresis system described above, on-chip detection or detecting or analyzing separated components within a chip, is problematic as excitation light is scattered or otherwise disrupted by the substrate material surrounding each of the micro-channels in the chip, since a substrate material such as plastic has a higher background fluorescence compared to glass.
One approach to solving the problem of on-chip detection, which has also been employed in capillary systems, is to detect separated or eluted sample components in a detection cell positioned adjacent to, but physically separate from, the chip or capillary system. This may be referred to as “off-chip” detection. The detection cell houses a polymer matrix through which the eluted sample components pass; however, there are no other structural components within the detection cell. The eluted sample components in this case simply traverse or pass through the polymer matrix from the inlet end of the detection cell to the outlet end. Since there are no other physical structures within the detection cell, excitation light that is directed into the detection cell only passes through the outer detection cell wall and through the polymer matrix rather than through multiple channel walls or capillary tube walls. This results in less scattering of the excitation light. The location within the detection cell where such excitation light is passed may be referred to as a “detection zone.”
The composition and configuration of the detection cell are selected to provide superior optical characteristics, e.g., a flat quartz chamber or low refractive index material. U.S. patent application Ser. No. 09/812,750, filed Mar. 19, 2001 and entitled “Detection Cell For Guiding Excitation Light Therein and Method For Using Same,” incorporated herein in its entirety by reference, is directed to such a detection cell that is made of a material having a low index of refraction lower than the polymer matrix to further reduce scattering by confining the excitation light between the walls of the detection cell having the lower index of refraction.
Maintaining the integrity of the eluted sample components in the polymer matrix of the detection cell, however, is important. In other words, as the eluted samples pass through the polymer matrix of a detection cell, the eluted sample components tend to distort or disperse in both vertical and horizontal directions, thereby reducing detection signal intensity and interfering with adjacent eluted sample components. Therefore, it is important to confine the eluted sample components to a “path” in the polymer matrix of the detection cell so that the eluted sample components will pass through the detection cell without significant dispersion.
In some capillary systems, such eluted sample components exiting the capillary are transported by a “sheath flow” of liquid through the detection cell to a detection zone within the detection cell where detection or analysis of the sample band takes place. A drawback of sheath flow systems is that in order to avoid distortion of a sample component, precise control of the flow rate of the sheath flow liquid is required. Another drawback of sheath flow systems is that the pressure used to drive the flow of the sheath flow liquid can cause back flow of the separation media in the channels, thereby impacting resolution. Further, the liquid flow that creates the “sheath” imparts a velocity profile to the eluted sample component, which, in turn, affects the index of refraction and the resolution.
In other off-chip detection systems, an eluted sample component (including an eluted sample component that is a discrete peak or band and an eluted sample component that elutes as a continuous stream) is transported from the outlet of the channels to a detection zone located in a detection cell by electrophoresis under the influence of the same voltage difference used to conduct the electrophoretic separation. Examples of capillary electrophoresis apparatus employing such detection systems are found, for example, in U.S. Pat. Nos. 5,529,679 by Takahashi et al and 5,583,826 by Nordman, both of which are incorporated herein in their entirety by reference. However, because of the larger cross-sectional area of the detection cell as compared to the lumen of the channels, the electric field diverges at each channel outlet causing a distortion of an eluted sample component. Such distortion results in severe loss of spatial resolution between subsequent sample components eluting from a single channel and between sample components eluting from adjacent channels. This loss of spatial resolution tends to reduce the detectability of these sample components and may result in actual mixing of the sample components or optical cross-talk between such sample zones.
FIG. 1 is a schematic top view of a portion of a microfluidic electrophoresis chip and illustrates the problem of distortion of sample components eluted from a microfluidic chip. FIG. 1 illustrates a microfluidic chip 100 having channels 104 through which charged sample components or bands 102 pass under a voltage applied between two electrodes 106, 108. As shown, discrete sample bands 102 are present within the channels 104 of the chip 100. Upon exiting the channels 104, however, the sample bands 102 are distorted while moving along their electrophoretic flow paths 110, as the sample bands 102 tend to follow the exemplary, divergent flow lines 112 (shown for one channel only).
Thus, there remains a need for an improved system and method for confining or otherwise reducing distortion of eluted sample components (including both eluted sample components that elute as discrete peaks or bands and those that elute as a continuous stream) passing from channels in a microfluidic chip or similar device.